Mouse organoid cultures more closely resemble in vivo cell organization than traditional cell cultures. In cancer research, organoids model the original tumor better than 2D cell lines and can be used to test potential cancer therapies in vitro to develop new drug treatments. For en bloc extraction of the male mouse urogenital system grasp the fat pad on either side of the bladder and pull upward to expose the testicle.
Carefully dissect each testicle away from the rest of the urogenital region. Gently lift the bladder so that the urogenital region is elevated together, exposing the urethra underneath. While holding the bladder, orient the scissors against the underside of the dorsal prostate to incise the urethra.
The entire urogenital region will be released from the abdominal cavity. After removing the urogenital system, the pelvic lymph nodes positioned immediately behind the urogenital system and on either side of the spine will be exposed. To remove the lymph nodes orient straight forceps under the lymph tissue and pull up.
Next, grasp the rectum with the straight forceps and cut, pulling up on the rectum to unravel the entire colon and small intestine to look for metastatic lesions in the mesenteric lymph nodes. When the entire ilium has been removed continue to pull on the duodenum to expose the stomach. Cut the esophagus to completely remove the stomach.
If any metastatic lesions in the lymph nodes are observed, carefully dissect these nodes away from the intestine for storage in PBS. When the stomach has been removed, the spleen can be harvested from the dorsal side of the abdomen for storage in 4%paraformaldehyde. Remove the liver for storage in PBS.
Remove the kidneys from either side of the spine along with any renal lymph nodes containing metastatic lesions. To expose the thoracic cavity carefully cut the diaphragm along the rib cage. The negative pressure released within the thoracic cavity will expose the heart and lungs.
Pull up on the sternum to open the thoracic cavity further. Scan the ventral face of the thoracic cavity along the rib cage for metastatic lesions in the thoracic lymph nodes for dissection if present. While still holding the sternum, cut away the ventral rib cage to access the heart and lungs and pull up on the heart to cut underneath the lungs.
To fully remove the heart and lungs en bloc cut all of the anterior blood vessels in the trachea. Carefully transfer the heart without damaging the lung tissue or lung metastatic lesions into a container of PBS. For dissection of the prostate tumor place the urogenital region under a dissection microscope.
Use a pair of straight forceps and a pair of curved forceps to flip the urogenital region onto its dorsal surface. Locate the proximal prostate region that can be identified by the pink-red color of the urethra. Grasp the urethra firmly to allow manipulation of the urogenital tissue with the curved forceps.
Locate the base of the seminal vesicles to allow their careful removal. Then use the curved side of the forceps to remove the vas deferens and as much fatty and connective tissue as possible. While still firmly holding the urethra and proximal prostate region, use a pair of fine pointed scissors to remove the bladder from the urethra.
Holding the proximal prostate region and urethra firmly with the straight forceps grasp the anterior prostate region with the curved side of the forceps to firmly pull the tissue away from the bladder and the rest of the prostate. Place the anterior prostate tissue in PBS and locate the ventral prostate region. Remove the ventral prostate region in the same manner.
If the lateral region can be distinguished from the dorsal region remove the lateral prostate region on both sides. Then remove the dorsal prostate region and place the proximal prostate region in 4%paraformaldehyde. After mincing, place the tumor pieces into a 15-milliliter tube of digestion buffer for a one-and-a-half to two hour digestion at 37 degrees Celsius.
At the end of the digestion, sediment the digested tissue by centrifugation and discard the supernatant. Flick the tube to loosen the cell pellet. Resuspend the cells in one milliliter of pre-warmed trypsin supplemented with 10 micromolar Y-27632 ROCK inhibitor for five minutes at 37 degrees Celsius.
At the end of the incubation triturate the tissue slurry five times with a standard P1000 pipette tip. Return the tube to the water bath for an additional five minute incubation and trituration. For matrix dome plating wash the digested cells in nine milliliters of cold medium, then collect the cells by centrifugation.
Resuspend the cells in one milliliter of fresh medium for counting. And after counting, collect the cells by centrifugation once again. Dilute the tumor cells in the appropriate volume of matrix.
Carefully drop 200 microliters of matrix cell solution into each well of a 55 degree Celsius warmed six-well plate. Allow the domes to solidify at room temperature for two minutes before placing the plate upside down in a 37 degrees Celsius incubator for 20 minutes. When the domes have fully solidified add two milliliters of prostate organoid medium supplemented with androgen R1881 and ROCK inhibitor to each well.
Return the plate to the cell culture incubator. Fluorescent dissection images reveal green fluorescent protein, or GFP expression, by solid prostate tumors, indicating that the tumor cells express Cre. The liver and lungs from the same animal exhibit metastatic tumors expressing GFP demonstrating that these tumors originated from the primary prostate tumor.
The pelvic lymph node from this mouse expresses GFP and not Tomato, indicating that this metastatic tumor has overtaken the lymph node and no normal tissue remains. At day one of culture small organoids generated from a solid prostate tumor begin to form with both Tomato and GFP-expressing cells present in the tumor organoid culture. By day seven and beyond when the prostate tumor organoids have fully formed however, the organoids express GFP, but not Tomato, suggesting that the organoids have originated from Cre-expressing tumor cells and not from normal epithelial cells.
On day one of culture of small organoids generated from fluid-filled prostate tumor both Tomato and GFP-expressing cells are present within the organoid culture. Organoids from fluid-filled prostate tumors express either GFP or Tomato at day seven and beyond however, indicating that the organoids formed from cells that do not express Cre. To stay oriented when harvesting the prostate tissue, be aware of the bladder and urethra positions at all times.
After their generation the organoids can be used in imaging applications, molecular biology analysis and drug screens.
